Project Summary This continuing research is aimed at characterizing, at micro- and nanoscales, the collective molecular events of cellular signaling that are initiated by plasma membrane receptors or ion channels and are highly orchestrated in space and time. Mast cells, which are stimulated by IgE-receptors and play a pivotal role in immune responses, serve as a valuable model system for investigating basic mechanisms by which cells respond to specific stimuli in a noisy environment. Proposed research will build on established thrusts: 1) Delineate with increasing resolution the initiation of stimulated cell signaling with a focus on critical but subtle events occurring at cellular membranes; 2) Translate the advanced technologies and refined hypotheses we have developed in model cells to disruption of cellular processes associated with neurodegenerative diseases. Innovative aspects include advancing an imaging modality of fluorescence correlation spectroscopy (ImFCS) to measure dynamic interactions of signaling-related components within the heterogeneous plasma membrane. The large data sets and robust analytics afforded by ImFCS yield precise values for multi-component diffusion and transient confinement parameters, revealing subtle interactions experienced by selective probes. This and other quantitative fluorescence microscopies will be used to evaluate constitutive and stimulated formation of kinase complexes, membrane trafficking, and mitochondrial dynamics, which are disrupted in cellular pathologies. Specific Aim 1 will implement ImFCS to measure diffusion properties of transmembrane proteins and proteins anchored to plasma membrane inner and outer leaflets to reveal distinctive environments experienced by these probes before and after antigen-crosslinking of IgE-receptors to initiate transmembrane signaling. We will measure stimulated changes in seconds timescale and collaboratively adapt theoretical models to interpret our results in terms of time-dependent changes of IgE-Fc?RI cluster size as related to early signaling events. Specific Aim 2 will collaboratively investigate model cells to gain mechanistic insight into cell- based pathologies associated with neurodegeneration. Building on established phenotypes, we will continue to evaluate structural features of ?-synuclein variants that disruptively modulate membrane trafficking and mitochondrial activities in Parkinson?s disease. We will use micro-patterned surfaces and ImFCS to yield new understanding of microglia-mediated hydrolysis of A? fibrils that goes awry in Alzheimer?s disease and phosphatidyl inositol kinase complexes that are disrupted in hypomyelination diseases.